The HIV Rev protein interacts with the viral IN in virus-infected cells. (A) The presence of Rev, IN and of actin (control) was detected using the appropriate specific antibodies and by western blots. (B) Co-IP of Rev and IN in cells infected by WT HIV-1, co-IP experiments were performed exactly as described in the Materials and methods section.

The appearance of the early Rev is due to de novo synthesis. (A) HeLa cells were infected with WT or ΔRev HIV-1 (MOI of 25) and immunostaining and observation of immunostained cells (arrows indicate the presence of Rev) as described in the Materials and methods section. (B) Treatment of cells by cycloheximide, actinomycin D and AZT as well as detection of Rev as described in the Materials and methods section.

The Rev–IN complexes can be dissociated by specific cell permeable peptides and stimulate integration. (A) H9 lymphocytes were incubated for 2 h with the INr-1 and INr-2 (Levin et al., 2009) peptides as well as with the Rev 13–23 and Rev 53–67 (Rosenbluh et al., 2007; Hayouka et al., 2008) peptides and then were infected by the WT HIV-1. Rev, IN and actin were precipitated from whole cell extract and were detected by western blot analysis using the appropriate antibodies as described in Fig. 1A. (B) 293T cells and Rev10 cells were incubated for 2 h with INr-1 (vertical strips and white bars, respectively), INr-2 (horizontal strips and gray bars, respectively) and with a mixture of both peptides (black squares and black bars, respectively) and then infected with the indicated viruses. Viral cDNA integration was estimated 24 h PI. (C) The following cell lines; H9 lymphocytes (white), SupT1 lymphocytes (gray), TZM-bl (horizontal strips), 293T (black squares) and Rev10 cells (black) were incubated with the indicated peptides for 2 h and then infected with MOI 1.0 WT HIV-1 and viral p24 was estimated at 72 h PI. (D) Exactly as (B) but total viral DNA was determined 12 h PI. (E) Rev10 cells were incubated with the INr-1 and 2 peptides for 2 h, infected with the WT HIV-1 and following co-IP, Rev, IN and actin were detected as described above in A. (B and D) P < 0.01, (C) P < 0.05. Designation of the different virus types as described in Materials and methods.

High degree of cDNA integration results in cell lysis. H9 lymphocytes were infected with increasing amounts (filled triangle, 0.01, 0.1 and 1.0 MOI) of the indicated HIVs. integration events/cell (A), cell death (B), p24 (C) and infectious particles (D) were estimated at the following different times (h) PI 12 (red), 24 (yellow), 60 (green), 108 (blue) and 156 (orange). (B) P < 0.01 and (A, C, D) P < 0.05.

A functional Rev or IN is not required for the formation of Rev–IN complex. Co-IP experiments were performed using the appropriate specific antibodies from lysate obtained cells infected by: (A) IN D64N D116N HIV-1, (B) Rev M10 HIV-1 or (C) ΔRev HIV-1 on Rev10⁺ cells, (D) ΔRev HIV-1. The presence of Rev, IN and actin was detected as described in (A). All other experimental details as described in the Material and methods section.

The HIV Rev protein inhibits IN-mediated integration and infection. The following cell lines; H9 lymphocytes (white bar), SupT1 lymphocytes (gray bar), TZM-bl (horizontal strips bar), 293T (black squares bar) and Rev10 cells (black bar) were infected by MOI 1.0 (A, C and E) and 2.0 (B and D) of the indicated HIVs and integration (A and B) as well as total viral cDNA (D and E) and viral p24 (C) were estimated at 24, 12 and 72 h PI, respectively. (A, B, D, E) P < 0.01, (C) P < 0.05. All others experimental conditions as well as the designation of the different virus types as described in the Materials and methods section.

Inhibition of integration by the Rev derived peptides. (A) Sup-T1 cells were incubated for 2 h with Rev 13–23 (white bars), Rev 53–67 (gray bars) as well as with both peptides (black bars) and were infected by the WT HIV-1 or by the DRev HIV-1. Viral cDNA integration was estimated 24 h PI. (B) Exactly as (A) but total viral DNA was determined 12 h PI, P < 0.01. Designation of the different virus types as described in Materials and methods.

High degree of cDNA integration results in cell lysis. Cell death was estimated 48 h PI (A) or 96 h PI (B) in peptide-treated H9 lymphocytes infected by ΔEnv HIV-1 or 48 h PI in peptide-treated H9 lymphocytes infected by Rev M10 (C) as well as at different times in cells infected by the WT virus (D). (E) as in (A) but peptides were at different times PI. (F) as in (A) but cells were double infected with ΔEnv HIV-1 (second infection was 12 h post first infection). (G) as in (A) but cells were infected by IN mut D64, 116N HIV-1. P < 0.05.